Viral diseases of crop plants cause significant yield and economic losses and this poses a major threat to global food security. To make matters worse there are no effective antiviral chemicals available and, although naturally resistant host genotypes exist, they are so rare that conventional breeding techniques cannot be used reliably to create resistant plants. The most effective option to combat phytopathogenic viruses is through biotechnological intervention, such as the use of genetic engineering to develop transgenic plants or the topical use of RNA silencing technologies to prevent or modulate the severity of the viral infection. Since the first report on the virus resistance of transgenic tobacco plants in 1986, enormous progress has been made in this field. In addition great strides have been made in our ability to genetically manipulate plants and viruses leading to a plethora of novel applications. This has prompted the need for this timely book which distills the most important research to provide a timely overview.

This authoritative book contains fifteen chapters whose breadth reflects the diversity of this research area. Topics covered range from: understanding the mechanisms of virus resistance in plants, and the management of whitefly-transmitted viruses, to the principles and methods involved in genetic engineering of virus resistant plants. Other chapters cover individual crops such as papaya, cassava, rice, tomato, and banana, for which virus resistance has been accomplished by employing different transgenic technologies.

This volume is essential reading for everyone working in this field, both students and specialists, from academia, research institutes/organizations and industries.

Considerable advances have been made in the past decade in understanding the mechanisms of virus resistance in plants. In particular, RNA silencing, resistance (R) protein-associated effector triggered immunity (ETI), and recessive resistance (impaired susceptibility), have been at the forefront. While conserved pathogen associated molecular patterns (PAMPs) have generally not been identified in viruses, it is highly likely that virus-encoded glycoproteins or proteins such as the coat protein, and ribonucleoprotein complexes are analogous to PAMPs and can trigger pathogen triggered immunity (PTI)-like responses. More recently, the role of the chloroplast, and its signalling with the nucleus and mitochondrion, has attracted attention in virus resistance studies. Developments in high throughput DNA and RNA sequencing, as well as protein and metabolite technologies, have enabled unravelling of defence responses to virus at a global level, leading to the current belief that resistance mechanisms form a complex of interacting networks. Unravelling these networks will provide opportunities for the next generation of new strategies for plant virus resistance engineering.

2. Role of host transcription factors in modulating defense response during plant-virus interaction

Plants are vulnerable to several environmental stresses either biotic or abiotic due to their sessile nature. Consequently, they assimilate various responses to counter and acclimate in ever-changing environments. One of the fascinating response is transcriptional reprogramming of cell leading to defense or stress adaptation. It is imperative to recognize transcription factors which are associated with plant defense responses against biotic agents such as viruses. Members of families belonging to WRKY family of transcription factor, myeloblastosis-related proteins (MYB), basic leucine zipper (bZIP), Apetela2/ethylene-responsive element binding (AP2/ERF), and NAC transcription factors have been shown to be associated with innumerable defense response against plant virus. These TF family members interact directly or indirectly to modulate defense response by activation or repression of downstream signaling pathways. Hence the gaining insight between plant virus and TFs interplay and deciphering the alterations in defense pathway are the prerequisite to engineer crops for tolerance to biotic stresses. In this chapter, we have attempted to summarize the explicit role of TFs in modulating the expression of defense genes during plant-virus interaction.

Epigenetics is a mechanism which determines the phenotype of an organism by causing heritable (during cell division) but simultaneously reversible alterations/variations in gene expression. It is not related to alterations in the DNA sequence of the genotype. Geminiviruses are the most devastating plant viruses since they cause significant yield losses in world agriculture. The plant defense initiated against these DNA viruses is of special interest, specifically in regard to the role of epigenetic mechanism played in control of virus spread. These heritable and covalent modifications of DNA and histone in virus genome are mainly related to suppression of gene transcription, despite the differences between viruses, the role of epigenetics seems to be reasonably comparable. However, several key questions remain unanswered concerning the basic mechanism behind the epigenetic regulation of viruses via plant defense system. This book chapter specifically summarizes the recent advances on role of epigenetics in virus genome modification leading to silencing of viral genes and plant tolerance/resistance.

4. Molecular markers as tools for identification and introgression of virus-resistant genes

A majority of the plant viral diseases are spread by insect vectors. Control of vectors using chemicals is a common practice for management of viral diseases. Although, to prevail such situations is still impractical as high recurring costs of the pesticides. Emergence of insecticide resistant insect populations, human anxiety regarding pesticide residue and its side effects also are other concern. Hence development of virus resistant crop plants is the need of future. In order to accelerate the current scenario of virus resistance breeding, molecular markers could function as a key tool to help and skip several generations of crossing and analysis altogether, thereby saving precious time. The researchers associated with the science of plant breeding and plant pathology has discovered reliable and rapid diagnosis techniques for many viral diseases using different molecular markers. Screening of viral disease resistance lines by marker-assisted selection (MAS) is most common technique, because phenotypic selection of virus resistant lines is always not convenient. It has a immense importance to come across the mandate of resistance breeding, hence markers like SCAR, RFLP, RAPD, SSR, AFLP, TRAP and CAPS known for their powerful genetic association could be used to supply complementary information to the classical genetic analyses. In resistance breeding programme gene tagging of particular trait is a necessary object for map based gene cloning and MAS. But mapping of genetic linkage is a time consuming procedure. In recent years, it is possible to identify markers directly without drawing any genetic linkage map. Markers based on nucleotide-binding and leucine-rich repeat domain (NB-LRR) is an advanced tool for identification of disease resistant genes. Markers based on molecular genetics can be used to determine the monogenic or polygenic disease resistance in crops which could provide durable resistance against wide range of pathogens. The gene pyramiding technique in crops is the best way for developing durable resistance (multigenic resistance) against multiple diseases. Based on molecular markers, identified segregating population in crops with viral disease resistance is needed to be confirmed against challenged viral inoculums either in controlled environment or in natural field conditions.

Viral diseases are a major menace for cultivation of crop plants across the globe. Non-availability of virucides and sources of host-plant resistance for their introgression in farmers preferred cultivars has put up major challenge for control of plant viral diseases. Hence genetic engineering for development of virus resistant transgenic crop plants is a promising option when compared to other conventional tools and techniques. Since the first report of virus resistant transgenic in 1986, significant progress has been made in understanding the molecular basis of virus resistance and also in the tools and techniques used for plant genetic engineering. Despite major advancement in the area of plant biotechnology, in the last 30 years, there has been no significant increase in the deployment of virus resistant transgenic crops, except for few examples such as papaya. Thus to help the plant biologists, here in this chapter we have compiled and briefly discuss all the tools and techniques available for engineering virus resistance in crop plants. Of all the strategies currently available for engineering virus resistance, RNA-interference-based technology has shown greater success. The recently developed genome editing technology has also given new hope for virus resistant transgenic crops. Without compromising the safety aspects of these transgenic crops the regulatory procedures followed in the approval of virus resistant transgenic crops for farmer's cultivation have to be refined to ensure that the benefit reaches to the famers without much delay.

6. Tools and techniques for production of double-stranded RNA and its application for management of plant viral diseases

Due to the rapidly growing global population, food production and security is the major challenge of agriculture. Plant viruses are obligate parasites that in some instances could cause up to 100% losses in a crop (e.g. maize streak disease). Although difficult to accurately determine the global economic impact that plant viruses have on agriculture, it is estimated that US$ 60 billion loss in crop yields worldwide each year is due to plant viral diseases. RNA silencing (RNA interference, RNAi) is a conserved endogenous pathway of all higher eukaryotes, which controls gene expression. RNAi is induced by double-stranded RNA (dsRNA) and allows the cell to recognize aberrant genetic material in a highly sequence-specific manner ultimately leading to its degradation, thus protecting the cell from subcellular pathogens, such as viruses and transposons. DsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses and against insects, vectors of plant viruses, via host induced gene silencing (HIGS). A non-transgenic approach employing RNAi has been used where enzymatically synthesized specific dsRNA molecules, when applied directly onto plant tissue, induce resistance against the cognate virus; as a result dsRNA molecules could be efficacious antiviral agents for crop protection. Next generation sequencing and bioinformatics analyses have provided a plethora of information and useful tools for the design and study of dsRNA application. In this chapter, the different methods for dsRNA production, both in vitro and in vivo, the means of direct application of the dsRNA molecules onto plants and several examples of non-transgenic dsRNA-mediated resistance are presented.

Viruses pose a major threat to worldwide production of papaya. Transgenic papaya varieties 'Rainbow' and 'SunUp' developed in the United States have provided durable resistance against local strains of Papaya ringspot virus (PRSV). Post-transcriptional gene silencing (PTGS) which exploits sequence homology between a transgene and the corresponding region of the invading viral genome has been used to successfully obtain PRSV-resistant plants in South America, the Caribbean, Asia, and Australia. Many of these transgenic lines await government and/or public approval prior to commercialization. This review re-emphasizes the success of Hawaiian PRSV-resistant transgenic papayas for sustainable virus resistance in the field, along with the availability of a proven transgenic toolkit. However emergence of new PRSV strains and mixed infections with viruses like Papaya leaf distortion mosaic virus and Papaya mosaic virus pose new challenges for future adoption of transgenic virus-resistant plants. In addition to PRSV, geminiviruses causing papaya leaf curl disease in Asia, Papaya meleira virus (and Papaya virus Q) in Brazil and Mexico are important targets. There is need to monitor field-level diversity and evolution of viruses against the backdrop of transgenic technologies available for next generation virus-resistant papaya benefitting farmers worldwide.

8. Development and delivery of transgenic virus-resistant cassava in East Africa

Cassava (Manihot esculenta) is a major staple food crop in sub-Saharan Africa where it provides food security and income to rural communities. The two virus diseases cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) constrain cassava production in these regions. RNA interference (RNAi) technology has been developed to address both diseases and has been shown to be effective in greenhouse and field studies. Field trials in Uganda and Kenya have demonstrated the potential of this technology to increase usable storage root yields greater than 50-fold under high CBSD pressure. This resistance is maintained across the cropping cycle and geographic locations from central Uganda to coastal Kenya, indicating its potential to help secure cassava production in East Africa. This review describes advances made in applying transgenic approaches to control CBSD and CMD and the challenges faced in delivering the improved planting materials to benefit cassava farmers in East Africa.

Rice is one of the world's most important crop plants. About sixteen viruses are known to infect rice worldwide. These viruses are diverse in nature and differ from each other in their molecular organization, vector transmission, symptoms produced and geographical distribution. Transgenic expression of viral coat proteins and nucleic acids has been an effective strategy for virus control in many plant species. The same methods have also been used for rice to engineer resistance against a majority of the infecting viruses. This chapter describes the genomic organization and gene functions of the viruses infecting rice and the transgenic resistance reported against them.

10. Whitefly-transmitted begomoviruses and advances in the control of their vectors

Whitefly-transmitted begomoviruses infect a large number of dicotyledonous hosts, causing many diseases of economic importance. Members of the genus Begomovirus are transmitted by only one whitefly species, Bemisia tabaci; and the symptoms include severe leaf curling, yellow mosaic, and yellow vein mosaic in several important agricultural crops. The insect vector B. tabaci plays a major role in the spread of many members of this virus group, and as a consequence, it also contributes to the emergence of new strains and species. Since the last decade, 360 begomovirus species and 41 morphologically indistinguishable B. tabaci species that were characterized across the globe have dramatically increased. Together, both the complex members of B. tabaci species and begomovirus species are spreading around the world; however, little information exists about the geographical distribution of this group of viruses, the relationships between its species, and the dependence for transmission on the only whitefly vector. The ability of multiple virus transmissions, the analysis of the B. tabaci species, and the identification of insect proteins implicated in virus replication and transmission will open up novel avenues for the management of this group of important diseases. This chapter provides an overview of the global diversity and geographical distribution of the B. tabaci species complex and their association with begomoviruses and recent advances in their control.

Virus resistant transgenic plants form an essential constituent of crop protection measures. Tomato is an important vegetable crop grown throughout the world for its nutritional benefits and tomato based processed food consumption. However, the production levels of tomato are threatened by many viral infections. In the absence of resistant tomato genotypes (where available genetic sources or resistance are scarce), development of transgenic resistance against pathogenic viruses is indispensable. The last couple of decades have witnessed substantial progress in incorporating virus resistance trait in tomato. This chapter provides overview of the strategies and successful instances of transgenic virus resistance with special emphasis on prominent viruses infecting tomato. Various approaches to incorporate virus resistance in tomato from antisense RNA expression, through various RNA interference (RNAi) based strategies and foray in to genome editing techniques are discussed. The significant achievements made in developing transgenic resistance to combat tomato leaf curl viruses, ground nut bud necrosis virus and cucumber mosaic virus are presented. Also, the utility of employing recently emerging genome editing tool in incorporating resistance to tomato viruses is also discussed.

Geminiviruses are plant-infecting, ssDNA containing viruses, which have twinned geminate particles. Majority of geminiviruses belongs to the genus Begomovirus having either monopartite or bipartite genome (DNA-A and DNA-B). Tomato leaf curl New Delhi virus (ToLCNDV), a pathogenic member of genus Begomovirus, causes Tomato leaf curl disease (ToLCD). ToLCD is responsible for economic losses of up to 100% in many regions of world. Control of ToLCV is very difficult as the classical vector control method turns out to be an unworthy option and Ty-based breeding yields partial success. Our laboratory has employed RNAi-based pathogen derived resistance (PDR) approach to successfully tackle ToLCD. We have overproduced artificial microRNA (a-miR) and artificial tasiRNA (a-tasiRNA) in tomato to silence the RNAi-suppressors encoded by ToLCVs. A few of the tomato transgenics show high level of virus resistance when challenged with broad range of ToLCVs.

Viral pathogens cause serious diseases in banana and plantains leading to a greater loss on the production and productivity. Banana Bunchy Top disease, Banana Streak, Banana Bract Mosaic and Infectious chlorosis or Banana mosaic pose serious concern in banana cultivation across the world. BBTV infection can result into 100% yield loss whereas other three diseases cause ca. 9 -70% yield loss. Breeding for virus disease resistance is not attempted probably owing to lack of resistance source, inherent problem in Musa due to sterility, parthenocarpic nature and compatibility. At present the management is mainly through timely identification of the diseased plants and eradication, and supply of certified virus free quality planting material to the farmers. Transgenic approaches have been attempted in a few labs across the world and came out with promising results for BBTV resistance but for other viruses attempts were not yet made. Transgenic resistance lines using RNAi has been developed for BBTV successfully but field level release and deployment in the farmers field is yet to take place. Multiple virus resistance targeting the suppressor of gene silencing with RNAi strategy is being attempted at ICAR-National Research Centre on Banana (ICAR-NRCB). CRISPR-Cas9 approach of genome editing for resistance and removal of eBSVs are the future goals of banana virologists to achieve the better management through molecular approaches. In this paper, we have reviewed and discussed about the important viruses of banana and transgenic management of them.

During the last two decades, virus-induced gene silencing (VIGS) has emerged as the most powerful and cost effective tool to determine gene functions in a relatively shorter period of time. VIGS has been used for both forward and reverse genetics studies for functional analysis of a large number of genes in both monocot and dicot plants. Various VIGS vectors have been developed, tested and modified to down-regulate the endogenous gene expression effectively in a sequence-specific manner in model plants, cereals, vegetables, fiber crops, fruits and timber trees. This review focuses on VIGS as a technique for inducing gene silencing in plants and its applications in understanding the role of genes controlling cellular and metabolic pathways, development, abiotic and biotic stress in economically important crop species.

15. Possible strategies for establishment of VIGS protocol in chickpea

Chickpea is the second largest legume in the world. The worldwide production of chickpea is far below its potential because of the factors like nitrogen deficiency, low nutrient absorption, flower or seed abortion and its vulnerability to the abiotic and biotic stresses. Consequently, it is important to understand the key molecular factors involved in stress tolerance, growth, flowering and seed development for the genetic improvement of the existing varieties. Currently the whole genome sequencing data and transcriptome information are widely facilitating functional genomic studies in chickpea. Further, marker based trait association mapping information is available for assistance in breeding program. However, information about exact function of genes is still lacking because of the absence of genetic mutants and difficult genetic transformation in this crop. Hence, the current scenario demands the establishment of virus-induced gene silencing (VIGS) technique in chickpea. VIGS would serve as an important tool for the functional characterization of large number of genes. Despite attempts by several research teams, the VIGS protocol is not yet available till date, though VIGS has been successfully applied for the gene characterization in other legumes. In this chapter we propose some strategies that can be attempted for development of successful VIGS protocol. We also describe our experience from present and past research projects aimed to study VIGS in chickpea.